Many plant storage tissues (seeds, leaves, roots, and tubers), accumulate sizable reserves of proteins during development. As an example, cultivated soybean seeds contain an average of about 40% protein, and in some varieties protein levels reach as much as 55% of the dry weight. The abundance of proteins in legume seeds has made them the primary dietary protein source and stimulated an interest in developing approaches to genetically engineer seeds to improve their nutritional quality. A related objective (as yet unrealized) is to utilize the protein synthesis and storage capacity of seed crops for the production of pharmacological or industrial proteins.
A major obstacle to improving the nutritional and functional qualities of plants has been the extremely low levels of accumulation of genetically modified or heterologous recombinant proteins that accumulate in plants transformed to express these proteins. For example, in experiments using comparable promoters, it has been shown that unmodified or very slightly altered seed storage proteins will accumulate in transgenic seed, but non-seed proteins or more highly modified seed storage proteins fail to accumulate (Hoffman et al. (1988) Plant Mol. Biol. 11:717-729; Jung et al. (1993) J. Exp. Bot. 44:343-349; Nielsen et al. (1995) J. Plant Physiol. 145:641-647; Saalbach et al. (1995) Mol. Breeding 1:245-258; Jung et al. (1998) Plant Cell 10:343-357). It has also been shown that antibodies fail to express in vacuoles (Frigerio et al (2000) Plant Physiol. 123:1483-1494) and that bovine β-casein fails to express in soybean seed vacuoles (Phillip et al. (2000) Annual Meeting for the American Society of Plant Physiology “Plant Biology 2000” Poster Abstract # 53, San Diego, Calif.)
Two of the most prevalent protein storage organs in plants are seed and paravienal mesophyll cells in leaves. Other plant storage tissues include tubers and roots. Storage proteins, especially those processed through the secretory pathway, generally undergo multiple post-translational processing steps including folding, assembly, intracellular sorting, and proteolytic processing, prior to final deposition (Müntz et al., (1993) Proc. Phytochem. Soc. Eur. 35:128-146; Müntz (1998) Plant Mol. Biol. 38:77-99; Herman and Larkins (1999) Plant Cell 11:601-613). The general mechanism of seed storage protein processing and deposition is highly conserved in dicot crop species including canola and soybean as well as monocot crop species including rice, wheat, and maize. Accumulation and deposition of the proteins is accomplished by compartmentalization in specialized vacuoles termed protein storage vacuoles and or protein bodies (Hara-Nishimura et al. (1995) J. Plant Physiol. 145:632-640; Müntz (1998) Plant Molec. Biol. 38:77-99; Herman and Larkins (1999) Plant Cell 11:601-613).
The proteolytic processing steps of protein deposition in vacuoles include specific polypeptide cleavage steps accomplished by proteases localized to the storage vacuole (Bassham et al. (2000) Curr. Opin. Cell Biol. 12:491-495). Storage proteins that accumulate in vacuoles have therefore co-evolved with the environment of the storage vacuole such that only a select few protease sites exist or are accessible to these proteases (Hara-Nishimura et al. (1987) Plant Physiol. 85:440-445; D'Hondt et al., (1993) J. Biol. Chem. 268:10884-10891; Hara-Nishimura et al. (1993) Plant Cell 5:1651-1659; Hara-Nishimura et al. (1995) J. Plant Physiol. 145:632-640).
The proteases that have thus far been implicated in the proteolytic processing of storage protein are the vacuolar processing enzyme family of cysteine proteases (also referred to as legumains), and specific aspartic proteases (Hara-Nishimura et al. (1987) Plant Physiol 85:440-445; D'Hondt et al. (1993) J. Biol. Chem. 268:20884-20891; Hara-Nishimura et al. (1993) Plant Cell 5:1651-1659; Hara-Nishimura et al. (1995) J. Plant Physiol. 145:632-640; Kinoshita et al. (1995) Plant Cell Physiol. 36:1555-1562; D'Hondt et al. (1997) Plant Molec. Biol. 33:187-192; Barrett et al., ed. (1998) Handbook of Proteolytic Enzymes, Academic Press, Sand Diego, pp746-749). In plants, vacuolar processing enzymes (VPE's) comprise a small gene family of plant asparaginyl endopeptidases implicated in the control of several important cellular process in addition to storage protein proteolysis. In Arabidopsis thaliana, three VPE's have been identified and are designated α-VPE, β-VPE, and γ-VPE. The genomic sequence of α-VPE is available as GenBank Accession No. AC004747 and the cDNA sequence at DDBJ Accession No. D61393; the genomic sequence of β-VPE is available as GenBank Accession No. AC007190 and the cDNA sequence at DDBJ Accession No. D61394; and the genomic sequence of γ-VPE is available as GenBank Accession No. ATF26P21 and the cDNA sequence at DDBJ Accession No. D61395. Two of these VPE's (α and γ) have been shown to be most abundant in vegetative tissue while the third (β-VPE) appears to be predominantly expressed in seed. The vegetative VPE's appear to be involved in protein turnover and mobilization of amino acid reserves in vegetative tissue during plant senescence process. β-VPE's localization and the results of in vitro processing suggest that β-VPE acts as the protease responsible for the cleavage and maturation of several major classes of seed storage proteins (Hara-Nishimura et al. (1991) FEBS Lett. 294:89-93).
Methods are needed to increase the accumulation of polypeptides of interest, both to enhance their quality and to harness the potential for the production of heterologous proteins in plant tissues.